This grant application requests continuation of the biosynthetic study of the antitumor alkaloids, camptothecin and perloline. The mechanistic details of their biosynthetic pathway are to be elucidated in vivo by examining the specific incorporation of radioactivity from putative complex carbon precursors. The results will define the structural and stereochemical prerequisites for the formation of these two alkaloids within the limits of the in vivo experimental system. A new approach to such investigations will be initiated and developed that will permit their biosynthesis to be studied in vitro using cell-free systems derived from plant tissue grown in cell culture. This application also requests support of the further study of camptothecin's molecular mechanism of action. Synthesis of three new types of camptothecin analogs and their evaluation as cytoxic and antitumor agents in vitro and in vivo will be done in attempts to prepare a clinically active antitumor drug based on the camptothecin pharmacophore. The effect these analogs and camptothecin have on purified DNA in vitro will be determined by assays designed to elucidate how such drugs cause DNA degradation in vivo.